CN110869515B - Sequencing method for genome rearrangement detection - Google Patents

Abstract

The present disclosure relates to single-end sequencing methods for improving genomic rearrangements such as deletions, insertions, inversions and translocations present in detection polynucleotides. The first priming event on the adapter allows sequencing of the target sequence, and the second priming event allows identification of sequences amplified and labeled by selective amplification. The combination of priming events in the same direction facilitates read alignment and identification of any genomic rearrangements.

Description

Sequencing methods for genomic rearrangement detection

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Non-Provisional Application No. 15/648,240, filed on July 12, 2017, which is hereby incorporated by reference in its entirety.

Field of the Invention

The present disclosure relates to sequencing methods, compositions and kits for improving the detection of genomic rearrangements, such as fusion genes. The present disclosure also relates to methods for preparing target polynucleotide libraries containing genomic rearrangements.

background

The ability to identify genomic rearrangements using nucleic acid sequencing methods has been shown to be very beneficial in the detection of human genetic disorders and diseases. Genomic rearrangement generally refers to any nucleotide rearrangement in a nucleic acid chain, including the deletion, insertion, inversion or translocation of one or more nucleotides, and can be detected by sequencing the nucleic acid of interest and comparing the sequence data with a reference (such as a known nucleic acid sequence). Next generation sequencing (NGS) can be used to quickly analyze polynucleotides and detect any genomic rearrangements in polynucleotides. NGS allows for simultaneous parallel analysis of a large number of sequences. In some forms, polynucleotides such as DNA are fixed on a solid surface by one or more adapters and amplified to increase signal intensity. Typically, a library for sequencing is prepared by fragmenting a sample into polynucleotide fragments, labeling the fragments with one or more adapters, and amplifying the polynucleotide fragments. Fragments can be amplified with one or more amplification primers. When sequencing by synthetic form, the fragments are hybridized with sequencing primers, and labeled dideoxynucleotides are added enzymatically. The signal from the labeled dideoxynucleotides is detected and analyzed to determine the sequence.

The polynucleotides of interest can be analyzed using single-end or double-end sequencing methods. The single-end sequencing method involves sequencing of genomic fragments from one end of the fragment to the other end. The single-end sequencing read provides a read for each fragment, and the one read corresponds to n base pairs at one of the two ends of the fragment, where n is the number of sequencing cycles. Single-end sequencing is generally not suitable for detecting large-scale genome rearrangements and repetitive sequence elements. The single-end reading across the fusion junction provides base pair evidence for the fusion event. However, it may be difficult to ensure that the single-end reading has been carried out to the number of base pairs sufficient to identify the fusion event.

The double-end method involves reading a nucleic acid fragment from one end to the other until a specified read length is reached, followed by another round of reading from the other side of the fragment. For the double-end method, a forward sequence read and a reverse sequence read are performed, and the data are paired as adjacent sequences. The sequence is matched to a reference sample to identify variants. Double-end sequencing methods are often used to detect genomic rearrangements because such methods generally provide good positioning information, making it easier to resolve structural rearrangements present in the genome. However, many sequencing instruments are not configured to perform double-end sequencing and are only capable of single-end sequencing.

WO 2007133831A2 discusses methods and compositions for obtaining nucleotide sequence information of a target sequence using adapters interspersed in a target polynucleotide. The method can be used to insert multiple adapters at spaced positions within a target polynucleotide or fragment. The adapter can be used as a platform for interrogating adjacent sequences using various sequencing chemistries, such as those that identify nucleotides by primer extension, probe ligation, etc. The present disclosure includes methods and compositions for inserting a known adapter sequence into a target sequence so that a continuous target sequence is interrupted by the adapter. The present disclosure points out that the identification of the entire target sequence can be completed by sequencing both the "upstream" and "downstream" of the adapter.

WO2015112974A1 discusses aspects related to methods for preparing and analyzing nucleic acids. In some embodiments, methods for preparing nucleic acids for sequence analysis (eg, using next generation sequencing) are provided.

WO2015148219A1 discusses a method for analyzing a target nucleic acid fragment, the method comprising using one strand of the target as a template to generate a first strand by primer extension using a first oligonucleotide primer, the first oligonucleotide primer comprising an overhang adapter region from 5' to 3', a primer ID region, a sequencing primer binding site, and a target-specific sequence region complementary to one end of the target fragment; optionally removing unbound primers; amplifying the target from the generated first strand to produce an amplified product; and detecting the amplified product. The disclosure also discusses why unique primers can be used in such a target analysis method.

Improved methods for detecting genomic rearrangements using single-end sequencing would be a useful contribution to the field, particularly if the approach is combined with high-throughput sequencing analysis.

SUMMARY OF THE INVENTION

Methods, compositions and kits for detecting genomic rearrangements in polynucleotides are provided. The methods, compositions and kits of the present invention can be used to more easily and reliably detect genomic rearrangements using single-end sequencing of nucleic acids of interest.

These and other features and advantages of the present invention will become apparent from the following detailed description, taken in conjunction with the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The present teachings will be best understood from the following detailed description when read in conjunction with the accompanying drawings.The features are not necessarily drawn to scale.

FIG. 1 shows one embodiment of a method for preparing polynucleotides for sequencing.

FIG. 2 shows another embodiment of a method for preparing a polynucleotide for sequencing.

Defined Terms

It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. The defined terms are in addition to the technical and scientific meanings of the defined terms as commonly understood and accepted in the technical field of the present teachings.

As used in the specification and the appended claims, in addition to their ordinary meanings, the terms "substantial" or "substantially" mean within the limits or degrees acceptable to those of ordinary skill in the art. For example, "substantially canceled" means that those skilled in the art consider the cancellation to be acceptable.

As used in the specification and the appended claims, in addition to their ordinary meanings, the terms "substantially" and "approximately" mean within limits or amounts acceptable to one of ordinary skill in the art. The term "approximately" generally refers to plus or minus 15% of the indicated value. For example, "about 10" may indicate a range of 8.7 to 1.15. For example, "substantially the same" means that one of ordinary skill in the art would consider the items being compared to be the same.

The terms "polynucleotide" and "nucleic acid" are used interchangeably herein to describe polymers of any length, such as nucleotides composed of greater than about 10 bases, greater than about 100 bases, greater than about 500 bases, greater than 1000 bases, up to about 10,000 or more bases, such as deoxyribonucleotides or ribonucleotides, or synthetically produced compounds (e.g., PNAs, as described in U.S. Pat. No. 5,948,902 and references cited therein), which can hybridize with naturally occurring nucleic acids in a sequence-specific manner, the manner being similar to the hybridization of two naturally occurring nucleic acids, such as can participate in Watson-Crick base pairing interactions. Naturally occurring nucleotides include guanine, cytosine, adenine, and thymine (G, C, A, and T, respectively). As used in the specification and the appended claims, unless otherwise indicated, a polynucleotide can be a polynucleotide with an adapter, a polynucleotide amplicon, or a polynucleotide amplicon with an adapter. A polynucleotide with an adapter differs from a polynucleotide of interest in that an adapter has been added to the polynucleotide of interest.

As used herein, the term "target nucleic acid" or "target" refers to a nucleic acid containing a target nucleic acid sequence. The target nucleic acid can be single-stranded or double-stranded, and is often double-stranded DNA. As used herein, "target nucleic acid sequence," "target sequence," or "target region" refers to a specific sequence or its complementary sequence. The target sequence can be in a nucleic acid in vitro or in vivo within the genome of a cell, and can be any form of a single-stranded or double-stranded nucleic acid.

"Hybridization" or "hybridizing" refers to the process by which fully or partially complementary nucleic acid chains come together under specified hybridization conditions to form a double-stranded structure or region, in which the two component chains are connected by hydrogen bonds. Although hydrogen bonds are generally formed between adenine and thymine or uracil (A and T or U) or between cytosine and guanine (C and G), other base pairs can also form hydrogen bonds (e.g., Adams et al., "The Biochemistry of the Nucleic Acids," 11th ed., 1992).

The term "primer" refers to an enzymatically prepared or synthesized oligonucleotide that can serve as a starting point for nucleic acid synthesis when forming a duplex with a polynucleotide template and extend from its 3' end along the template to form an extended duplex. The nucleotide sequence added during the extension process is determined by the sequence of the template polynucleotide. Primers are used as starting points for nucleotide polymerization catalyzed by DNA polymerase, RNA polymerase or reverse transcriptase. The length of a primer can be 4-1000 bases or longer, for example 10-500 bases.

As used herein, the term "primer extension" refers to extending a primer by annealing a specific oligonucleotide to a primer using a polymerase. The term "adapter" refers to a nucleic acid molecule attached to a polynucleotide of interest to form a synthetic polynucleotide. An adaptor may be single-stranded or double-stranded, and may include DNA, RNA, and/or artificial nucleotides. An adaptor may be located at the end of a polynucleotide of interest, or may be located in the middle or inside. An adaptor may add one or more functions or characteristics to a polynucleotide of interest, such as providing a priming site for amplification or sequencing or adding a barcode. For example, an adaptor may include a universal primer and/or a universal priming site, including a priming site for sequencing. As a further example, an adaptor may contain one or more barcodes of various types or for various purposes, such as a molecular barcode, a sample barcode, and/or a target-specific barcode. Various adaptors are known in the art and may be used in the methods, compositions, and kits of the present invention or may be used after modification. For example, an adaptor includes a Y adaptor, which may be attached to a polynucleotide to produce a library with a variable 5' end. Adaptors may also include separate sequences (e.g., A/B adaptors), wherein the A adaptor is attached to one end of a polynucleotide, and the B adaptor is attached to the other end of the polynucleotide. Adaptors also include stem-loop adaptors, wherein a hairpin loop is attached to the end of a polynucleotide; a portion (usually a stem) may be cut before amplification or sequencing. Adaptors may be attached to the polynucleotide of interest by any suitable technique, including but not limited to connection, the use of a transposase, hybridization, and/or primer extension. For example, an adaptor may be connected to the end of a polynucleotide of interest. As another example, an adaptor is attached by inserting a transposon comprising an adaptor into a polynucleotide of interest using a transposase, thereby providing an adaptor at the end of a fragment of a polynucleotide of interest. In some embodiments, an adaptor includes a target-specific primer and a target-specific barcode, which allows the adaptor to be attached to a polynucleotide of interest (more specifically, to a complementary polynucleotide) by primer extension of a target-specific primer.

The term "sequencing" refers to determining the identity of one or more nucleotides, ie, whether the nucleotide is G, A, T, or C.

The term "single-end sequencing" refers to the use of reading ("single-end reading") from one end of a polynucleotide to determine the sequence of a polynucleotide. Single-end reading can be performed by any sequencing process, including next-generation sequencing and other large-scale parallel sequencing technologies. Instruments configured to perform single-end sequencing are commercially available from many companies. For example, Illumina's HiSeq 2500 can provide a read length of 50bp and 100bp on a single end. In some embodiments, the nominal, average, mean or absolute length of a single-end read is at least 20 continuous nucleotides, or at least 30 continuous nucleotides, or at least 40 continuous nucleotides, or at least 50 continuous nucleotides. In some embodiments, the nominal, average, mean or absolute length of a single-end read is at most 300 continuous nucleotides, at most 200 continuous nucleotides, or at most 150 continuous nucleotides, or at most 120 continuous nucleotides, or at most 100 continuous nucleotides. The aforementioned minimum and maximum values can be combined to form a certain range.

As used herein, the term "portion" or "fragment" of a sequence refers to any portion of a sequence (e.g., a nucleotide subsequence or an amino acid subsequence) that is smaller than the entire sequence. The length of a portion of a polynucleotide can be any length, for example, at least 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200, 300, or 500 or more nucleotides in length. A portion of a guide sequence can be about 50%, 40%, 30%, 20%, 10% of a guide sequence, for example, one-third of a guide sequence or less, for example, 7, 6, 5, 4, 3, or 2 nucleotides in length.

The term "fusion gene" refers to a polynucleotide formed by two previously separated genes. Fusion genes can be produced by translocation, interstitial deletion or chromosomal inversion, which often occur in human cancer cells. Fusion genes can lead to the expression of fusion transcripts, which are translated into fusion proteins that change the normal regulatory pathways of cells and/or promote the growth of cancer cells. Gene variants may also produce abnormal proteins that affect normal regulatory pathways. Many fusion gene polynucleotides are known, and more are being discovered. For example, US20100279890, US20140120540, US20140272956 and US20140315199 disclose many fusion genes associated with cancer and other diseases, and methods for detecting such fusion genes. The methods, compositions and kits of the present invention can be used to detect known gene fusions, and can be used to find previously unknown gene fusions.

As used herein, the term "priming site" refers to a site within an oligonucleotide or polynucleotide that is configured to hybridize with a primer so that adjacent sequences or sequences close enough for single-end sequencing can be amplified or sequenced, for example, by primer extension. The priming site can be a sequence present in the polynucleotide of interest or a sequence added to the polynucleotide by adding an adapter that contains the priming site. An adapter containing a priming site can be added by ligation, by using a transposase, by primer extension, or by other techniques.

In the present disclosure, numerical ranges are inclusive of the numbers defining the range. In the present disclosure, wherever the word "comprising" is seen, it is contemplated that the phrase "consisting essentially of" or "consisting of" may be used instead. It should be recognized that chemical structures and formulae may be extended or expanded for illustrative purposes.

As used in the specification and the appended claims, the terms "a", "an", and "the" include singular and plural referents unless the context clearly indicates otherwise. Thus, for example, "a primer" includes a primer and a plurality of primers. In the present disclosure, ordinal numbers such as the terms first, second, third, etc. do not indicate that the first event occurs before the second event (unless the context indicates otherwise); rather, they are used to distinguish different events from each other.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

As disclosed herein, many numerical ranges are provided.It should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed with one-tenth of the unit of the lower limit as an interval (unless the context clearly indicates otherwise).Each smaller range between any stated value or intermediate value in the stated range and any other stated value or intermediate value in the stated range is included within the scope of the present invention.The upper and lower limits of these smaller ranges can be independently included or excluded in the scope, and each range in which any one, neither, or both of the limit values are included in these smaller ranges is also included within the scope of the present invention, subject to any clearly excluded limit value in the stated range.When the stated range includes one or both of the limit values, the scope excluding any one or both of these included limit values is also included in the present invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present teachings, some exemplary methods and materials are currently described.

All patents and publications mentioned herein are expressly incorporated herein by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present claims are not entitled to antedate such publication. In addition, the publication dates provided may be different from the actual publication dates that can be independently verified.

It will be apparent to those skilled in the art after reading this disclosure that each individual embodiment described and illustrated herein has discrete components and features that can be readily separated or combined with the features of any other several embodiments without departing from the scope or spirit of the present teachings. Any recited method can be performed in the order of events recited or in any other order that is logically possible.

DETAILED DESCRIPTION OF THE INVENTION

In some embodiments, the present disclosure provides a method for preparing a polynucleotide for sequencing by attaching a target-specific barcode. The method includes amplifying the polynucleotide with a first amplification primer and a second amplification primer, wherein the first amplification primer comprises a first priming sequence and a target-specific barcode, wherein the first priming sequence hybridizes to a first priming site of the polynucleotide. This amplification produces a polynucleotide amplicon, wherein the polynucleotide amplicon comprises a sequence identical or complementary to the polynucleotide of interest and the target-specific barcode.

The first amplification primer comprises a target-specific (i.e., complementary and/or hybridizing to a target sequence within a polynucleotide with an adapter) first amplification primer. The first amplification primer further comprises a target-specific barcode, which is a barcode specific to a target sequence, for example, a barcode specific to a portion of a gene (such as a portion of a fusion gene). The amplification produces a polynucleotide amplicon, wherein the polynucleotide amplicon comprises a sequence identical or complementary to the polynucleotide of interest and the target-specific barcode. The second amplification primer (1) hybridizes with a portion of an adapter attached to the polynucleotide at a distance from the first priming site, or (2) hybridizes with a second priming site of the polynucleotide, wherein the second priming site is at a distance from the first priming site. In some embodiments, the method may further include attaching an adapter to the polynucleotide to form an adapted polynucleotide, wherein the adapter comprises a second priming site and an optional adapter barcode. In some embodiments, the second priming site is on the adapter and is a universal priming site and/or a site for a sequencing primer, and/or the second primer binding site is a universal priming site at the 5' end of the polynucleotide with the adapter. In some embodiments, the adapter and/or the second priming site is at the 5' end of the chain of the polynucleotide, and the first priming site is at the 3' end of the chain. In some embodiments, the adapter barcode is a sample barcode or a molecular barcode. The molecular barcode can be a unique sequence because it is unique within a set of adapters attached to a pool of polynucleotides of interest.

In some embodiments, the present disclosure provides methods, compositions and kits for preparing a polynucleotide library for sequencing by attaching a target-specific barcode. A polynucleotide pool is amplified using a first set of amplification primers and a second set of amplification primers, wherein the first set of amplification primers hybridizes with a plurality of different sequences in the polynucleotide pool, wherein each of the first set of amplification primers comprises a different target-specific barcode. In some embodiments, an adapter comprising an adapter barcode is attached to a polynucleotide amplicon. The second set of amplification primers (1) hybridizes with a portion of an adapter attached to a polynucleotide at a distance from a first priming site, or (2) hybridizes with a second priming site of a polynucleotide, wherein the second priming site is at a distance from the first priming site. A polynucleotide amplicon library is generated by amplifying the first and second sets of primers. An adapter can be added to a polynucleotide amplicon. In some embodiments, an adapter is added before amplification, and the adapter comprises a second priming site hybridized with a second set of amplification primers. In some embodiments, an adapter is added after amplification, for example, so as to provide a sequencing priming site on a polynucleotide amplicon.

Each of the plurality of polynucleotide amplicons can be sequenced at two locations by performing a first primer extension and a second primer extension, wherein the first primer extension and the second primer extension sequencing are performed in the same direction for each of the polynucleotide amplicons with adapters. Genomic rearrangements can be identified based on data generated from the first primer extension and the second primer extension sequencing.

In other embodiments, the present disclosure provides a composition and a kit for detecting a genomic rearrangement in a polynucleotide having a first binding site. The composition and the kit include a first and a second amplification primer. The first amplification primer includes a target-specific primer and a target-specific barcode. The composition and the kit may further include an adapter. The adapter includes a second priming site and an adapter barcode. In some embodiments, the second amplification primer includes a priming sequence complementary to or identical to a sequence in the adapter, such as a second priming site. In some embodiments of the composition and the kit, the second amplification primer (1) hybridizes with a portion of the adapter attached to the polynucleotide at a distance from the first priming site, or (2) hybridizes with the second priming site of the polynucleotide, wherein the second priming site is at a distance from the first priming site. In some embodiments, the adapter and/or the second priming site is at the 5' end of the chain of the polynucleotide, and the first priming site is at the 3' end of the chain.

In other embodiments, the present disclosure provides methods, compositions and kits for detecting genomic rearrangements in polynucleotides. The methods, compositions and kits include amplifying a polynucleotide with a first amplification primer and a second amplification primer. The first amplification primer hybridizes with a first priming site of the polynucleotide, and the first amplification primer further comprises a target-specific barcode. The amplification produces a polynucleotide amplicon comprising a sequence identical or complementary to the polynucleotide of interest and the target-specific barcode. The polynucleotide amplicon is sequenced at the first and second positions by performing a first primer extension and a second primer extension. The first primer extension and the second primer extension can be performed in the same direction.

In the aforementioned methods, compositions and kits, the target-specific barcode is specific for a target, such as a gene, a portion of a gene, a fusion gene, a portion of a fusion gene, or other polynucleotide of interest. The fusion gene can be a known fusion gene, including a junction of a known fusion gene, and/or the fusion gene can be a suspected or hypothetical fusion gene, or a junction of such a fusion gene. The target can be a genomic rearrangement, such as a deletion, insertion, inversion, and translocation in a polynucleotide of interest. In some embodiments, the target is a cDNA junction or an exon junction.

In some embodiments, the second amplification primer hybridizes to a portion of the adaptor, such as a second priming site, which can be a sequencing priming site of the adaptor. In some embodiments, the adaptor-bearing polynucleotide comprises an adaptor at the 5' end and/or a target-specific barcode at the 3' end.

In some embodiments, the polynucleotide of interest comprises a plurality of polynucleotides of interest, and the method comprises attaching a plurality of adaptors to the plurality of polynucleotides, thereby forming a plurality of adaptor-bearing polynucleotides, each comprising a different adaptor barcode. Alternatively or additionally, wherein the polynucleotide of interest comprises a plurality of polynucleotides of interest, and the first amplification primer comprises a plurality of first amplification primers having different target-specific primers and target-specific barcodes, thereby forming a plurality of adaptor-bearing polynucleotide amplicons, each comprising a different target-specific barcode.

In some embodiments, the polynucleotide amplicon with the adapter is sequenced at the first and second positions by performing a first primer extension and a second primer extension, wherein the first primer extension and the second primer extension are performed in the same direction. In some embodiments, the first primer extension is performed with a first sequencing primer that is complementary or identical to a portion of the adapter, such as the second priming site. In some embodiments, the second primer extension is performed with a second sequencing primer that is complementary or identical to a portion of the first amplification primer, such as a portion that is adjacent to or close enough to the target-specific barcode for single-end sequencing of the target-specific barcode.

Sequencing by primer extension is performed as follows: primers are hybridized to polynucleotide amplicons; primers are extended by adding one or more labeled nucleotides, thereby producing incorporated labeled nucleotides; and the incorporated labeled nucleotides are detected. The sequencing primers may be complementary or identical to the sequences on the adapters. In some embodiments, the first primer extension and the second primer extension are performed in the same direction on the polynucleotides in separate sequencing runs. In some embodiments, sequencing is next generation sequencing (NGS) or massively parallel sequencing. The data generated by sequencing from the first primer extension and/or the second primer extension can be compared with known nucleic acid sequences (such as known gDNA sequences).

The methods, compositions and kits of the present invention can be used for sequencing polynucleotides, including genomic DNA (gDNA), complementary DNA (cDNA) derived from RNA templates (e.g., messenger RNA (mRNA) or microRNA (microRNA)), mitochondrial DNA (mtDNA), RNA (e.g., mRNA, microRNA) and other polynucleotides. The polynucleotides can be of any origin, such as microorganisms, viruses, fungi, plants or mammals.

In some embodiments, the methods, compositions and kits of the present invention are used to detect the presence, position or absence of genomic rearrangements in polynucleotides of interest. Genomic rearrangements can be deletions, duplications, insertions, inversions or translocations, and the methods, compositions and kits can be used to detect whether certain genomic sequences or genes have been deleted, repeated, inserted, inverted or translocated in the polynucleotides of interest. In some embodiments, the methods, compositions and kits of the present invention are used to detect genomic deletions. In some embodiments, the methods, compositions and kits of the present invention are used to detect genomic duplications. In some embodiments, the methods, compositions and kits of the present invention are used to detect genomic insertions. In some embodiments, the methods, compositions and kits of the present invention are used to detect genomic inversions. In some embodiments, the methods, compositions and kits of the present invention are used to detect genomic translocations. In some embodiments, the methods, compositions and kits of the present invention are used to detect genomic rearrangements in polynucleotides such as gDNA or cDNA derived from RNA. In some embodiments, the frequency of genomic rearrangements is about 100% or less, or about 50% or less, or about 10% or less, or about 5% or less, or about 1% or less. In some embodiments, the method further comprises detecting genomic rearrangements using single-end sequencing of the polynucleotide amplicon, for example by identifying genomic rearrangements based on data generated from sequencing of the first primer extension and the second primer extension. In some embodiments, the genomic rearrangement is a translocation.

A sequencing method that can be used to detect genomic rearrangements in polynucleotides is provided. The method of the present invention can be used to more easily and reliably detect genomic rearrangements using single-end sequencing of nucleic acids of interest. The method of the present invention can be used in the next generation sequencing (NGS) process to detect deletions, insertions, inversions and translocations in polynucleotides of interest. The method of the present invention involves sequencing of first and second primer extensions in the same direction to improve the accuracy of polynucleotide rearrangement detection. The combined sequence data from the first and second primer extensions contributes to the alignment of reads in the polynucleotides and the identification of genomic rearrangements. The combination of reads generated in the same direction allows for more accurate identification of the relative position of nucleic acids in polynucleotides. Compared with standard single-end sequencing methods, the method of the present invention improves the ability of the single-end sequencing process to identify the relative position of nucleotides in the genome, thereby producing a more effective structural rearrangement analysis.

The method of the present invention can be used in high-throughput sequencing methods, such as next-generation sequencing (NGS) processes. In some embodiments, the high-throughput sequencing method includes three steps: preparation, fixation and sequencing of the library. Polynucleotides are usually randomly fragmented, and adapters are connected to one or both ends of the fragments. The adapters can be linear adapters, circular adapters or bubble adapters. The sequencing library fragments are fixed on a solid support, and parallel sequencing reactions are performed to inquire about the polynucleotide sequence. High-throughput sequencing methods can use emulsion PCR, bridge PCR or rolling circle amplification to provide copies of the original polynucleotides.

Polymerases tend to make errors during PCR (most commonly misincorporation of nucleotides), and if these errors occur in early cycles, they appear as variants in sequencing data analysis. Molecular barcodes can be used to distinguish PCR errors from actual variants in the polynucleotides of interest. The concept of molecular barcodes is that each polynucleotide in the library to be amplified is attached to a unique molecular barcode. Sequence reads with different molecular barcodes represent different original DNA molecules, while reads with the same barcode are the result of PCR replication from the same original molecule. Molecular barcodes called degenerate base regions (DBRs) are disclosed in U.S. Patent 8,481,292 (Population Genetics Technologies Ltd.). DBRs are random sequence tags attached to molecules present in a sample. DBRs and other molecular barcodes allow PCR errors in sample preparation to be distinguished from mutations and other variants present in the original polynucleotides.

Attaching adaptors to polynucleotides

In some embodiments, polynucleotides are attached to adapters to form polynucleotides with adapters. Adapters can be attached to polynucleotides before or after amplification, in some embodiments, polynucleotides are polynucleotide amplicon, and polynucleotides with adapters are polynucleotide amplicon with adapters. Adapters can be attached by any suitable technology, such as by connection, use of transposase, hybridization and/or primer extension. In some embodiments, polynucleotides are connected to adapters at one or both ends. In ligation, covalent bonds or connections are formed between the ends of two or more polynucleotides (such as nucleic acids of interest) or oligonucleotides (such as adapters). The properties of the bonds or connections can vary, and can be connected enzymatically or chemically. Usually connected enzymatically, to form a phosphodiester bond between the 5' carbon of the terminal nucleotide of a polynucleotide or oligonucleotide and the 3' carbon of another polynucleotide or oligonucleotide. In some embodiments, the adaptor is a Y adaptor, which can generate libraries with varying 5' ends and with P5 and P7 priming sites suitable for use on MiniSeq, NextSeq, and HiSeq 3000/4000 sequencing instruments.

In some embodiments, A/B adapters are attached to the polynucleotides of interest, wherein the A adapter is attached to one end of the polynucleotide, and the B adapter is attached to the other end of the polynucleotide. In some embodiments, the A/B adapters are attached by random connection or using a transposase or by primer extension for amplification. It can be expected that the individual characteristics of the A adapters and the B adapters provide that each polynucleotide included in the sequencing program will include both A and B adapters (i.e., one type of adapter is attached to the 5' end of each polynucleotide undergoing sequencing, and another type of adapter is attached to the 3' end, expressed as an A/B adapter combination). Due to the random nature of the connection step, polynucleotides with A/A and B/B adapters will also be generated, and subsequent processing steps can be used to ensure that only molecules with A/B adapter combinations are selected and/or included in the sequencing program. Polynucleotides with adapters can be amplified using primers for the part of the adapters before or after the amplification for attaching target-specific barcodes as described herein to increase the amount of the polynucleotides of interest. In some embodiments, adaptors are ligated in a pattern and to a sufficient number of polynucleotides to generate a fully sequenceable library for massively parallel sequencing.

In some embodiments, the adapter comprises an adapter barcode. The adapter barcode can be used as any desired purpose, such as an identifier of the source or property of a polynucleotide. A barcode generally refers to any sequence information used for polynucleotide identification, grouping or processing. A barcode can be included to identify a single read, a read group, a subset of reads associated with a probe, a subset of reads associated with an exon, a subset of reads associated with a sample or any other group, or any combination thereof. For example, a sequence can be sorted (e.g., using a computer processor) by a sample, an exon, a probe group, or a combination thereof with reference to the barcode information. The barcode information can be used to assemble overlapping groups. A computer processor can recognize barcodes and assemble reads by organizing the barcodes together.

Polynucleotides can be obtained by any suitable mechanism. The polynucleotides of interest can be genomic deoxyribonucleic acid (gDNA), cDNA, mRNA, mitochondrial DNA or other types. Polynucleotides can be mammalian, viral, fungal or bacterial polynucleotides or mixtures thereof. In some embodiments, before the adapter is attached to the polynucleotide, any suitable technology is used to make polynucleotide chains such as genomic DNA fragmentation. As known in the art, physical fragmentation, enzymatic fragmentation or chemical shearing fragmentation can be used to fragment polynucleotide chains. In some embodiments, physical fragmentation methods such as ultrasonic treatment, acoustic shearing or fluid dynamic shearing are used to fragment polynucleotides. In some embodiments, restriction enzymes are used to fragment polynucleotides. In some embodiments, enzymes such as DNase I or transposase are used to fragment polynucleotides. In some embodiments, chemical shearing methods such as thermal digestion are used in the presence of metal cations to fragment polynucleotides. In some embodiments, polynucleotides are randomly fragmented. In some embodiments, polynucleotides can be treated with sodium bisulfite or other chemical modifiers. In some embodiments, polynucleotide fragments are used to fill (populate) sequencing libraries.

The polynucleotide fragments can have any suitable base length. In some embodiments, the polynucleotide fragments have a base length of about 30 to about 2,000. In some embodiments, the polynucleotide fragments have a base length of about 30 to about 800. In some embodiments, the polynucleotide fragments have a base length of about 30 to about 500. In some embodiments, the polynucleotide fragments have a base length of about 100 to about 800. In some embodiments, the polynucleotide fragments have a base length of about 200 to about 600.

After fragmentation, one or more adapters can be attached to the polynucleotide fragments. In some embodiments, the adapter is a linear adapter, a circular adapter, or a bubble adapter. In some embodiments, the polynucleotide is connected to at least one circular adapter. In some embodiments, the polynucleotide fragments are contacted with the circular adapter to generate circular polynucleotide molecules. In some embodiments, only the circular polynucleotide molecules are amplified during the amplification process. In any of these embodiments, the adapter may include an adapter barcode.

Amplification of target polynucleotides

The method of the present invention includes amplifying the polynucleotide before and/or after the polynucleotide is attached to the adapter. In some embodiments, the adapter is located at the 5' end of the sequence of interest in the polynucleotide, and the adapter provides a priming site for amplifying the sequence of interest. The polynucleotide with adapters is amplified using the first amplification primer and the second amplification primer. The first amplification primer has sequence specificity to the target sequence in the polynucleotide and can hybridize with a part of the target sequence (polynucleotide of interest). The second amplification primer can hybridize with the priming site of the adapter or with the target-specific priming site of the polynucleotide of interest. During the amplification step, the first amplification primer hybridizes with the target sequence, and the second primer hybridizes with the sequence priming site on the adapter. In some embodiments, the first amplification primer hybridizes at the 5' end of the polynucleotide with adapters. The primer of the method of the present invention should be large enough to provide sufficient hybridization with the target sequence of the polynucleotide.

In order to amplify, the polynucleotide of interest is hybridized with the first amplification primer comprising a target-specific barcode. The first amplification primer is complementary to at least a portion of the polynucleotide. The first amplification primer is hybridized with the first priming site of the polynucleotide. The polynucleotide comprises a target sequence at the 3' end, optionally followed by an adapter. If the target sequence is present in a polynucleotide with an adapter, the first amplification primer is hybridized with the polynucleotide with an adapter, thereby allowing selective amplification and detection of the target sequence. The first amplification primer can be complementary and/or hybridized with a genomic rearrangement, such as a deletion, insertion, inversion or translocation in the polynucleotide of interest. In some embodiments, the first amplification primer is complementary and/or hybridized with a cDNA junction or an exon junction. In some embodiments, the first amplification primer is complementary and/or hybridized with a fusion gene, such as a known fusion gene, including a junction of a known fusion gene and/or a suspected or hypothetical fusion gene or a suspected or hypothetical fusion gene.

The second amplification primer hybridizes to the polynucleotide or adapter at a distance from the first priming site. In some embodiments, the second amplification primer hybridizes to a portion of the adapter attached to the polynucleotide at a distance from the first priming site. In some embodiments, the second amplification primer hybridizes to a second priming site of the polynucleotide, wherein the second priming site is at a distance from the first priming site.

Any suitable method can be used to increase the polynucleotide of interest. In some embodiments, the polymerase chain reaction (PCR) is used to amplify polynucleotides. Usually, PCR includes the denaturation (such as DNA melting) of polynucleotide chains, the annealing of primers to the polynucleotide chains of denaturation, and the polynucleotides of synthetic complementation with polymerase extension primers. The process usually requires DNA polymerase, forward and reverse primers, deoxynucleoside triphosphates, divalent cations and buffer solutions. In some embodiments, by linear amplification, polynucleotides are amplified. In some embodiments, emulsion PCR, bridge PCR or rolling circle amplification are used to amplify polynucleotides. The polynucleotides amplified can be analyzed using a suitable sequencing method to determine the order of base pairs.

In some embodiments, one or more primers or polynucleotides are fixed on a solid support. The immobilization of amplification primers and/or polynucleotides can promote the washing of polynucleotides to remove any undesirable species (e.g., deoxynucleotides). In some embodiments, polynucleotides include one or more adapters attached to a solid support, so that polynucleotides are fixed on a support. In some embodiments, polynucleotides are fixed on the surface of a flow cell or a slide. In some embodiments, polynucleotides are fixed on microtiter wells or magnetic beads. In some embodiments, a polymer-coated solid support attached to a functional group or module can be used. In some embodiments, a solid support can be provided with a functional group, such as an amino group, a hydroxyl group, or a carboxyl group, or other modules, such as avidin or streptavidin, for attaching an adapter.

The polynucleotide amplicon can be a polynucleotide amplicon with an adapter. In some embodiments, the polynucleotide or polynucleotide amplicon with an adapter comprises a binding partner, such as a biotin module. The polynucleotide can be attached to an adapter comprising a binding partner, or the polynucleotide can be amplified using one or more primers comprising a binding partner. In some embodiments, the method of the present invention includes forming a complex between mutual binding partners, such as biotinylated primer extension products and anti-avidin or streptavidin supported by a solid. The method may also include enriching a sample containing a polynucleotide with an adapter containing a binding partner by binding to the mutual binding partner. Avidin and streptavidin proteins form abnormally tight complexes with biotin and certain biotin analogs. Typically, when biotin is coupled to a second molecule through its carboxyl side chain, the resulting conjugate is still tightly bound by avidin or streptavidin. When such a conjugate is prepared, the second molecule is said to be "biotinylated". Typically, the method of the present invention involves compounding a biotinylated nucleic acid with avidin or streptavidin, and then detecting, analyzing and/or using the complex. In some embodiments, biotinylated polynucleotides are fixed on a flow cell coated with streptavidin or on a metal bead coated with streptavidin. In some embodiments of the methods, compositions and kits of the present invention, a target-specific primer (e.g., a first amplification primer) can be attached to a binding partner, such as a biotin module, to allow selection or purification by binding to a mutual binding partner such as streptavidin or avidin. Useful binding partners include biotin: avidin, biotin: streptavidin, antibody: antigen and complementary nucleic acid. In some embodiments, a target-specific primer may include a binding partner, such as biotin, to allow capture of a selectively amplified pool.

For the preparation of polynucleotides for next-generation sequencing, target enrichment is generally employed prior to next-generation sequencing, and one or more target enrichment schemes may be included in the present method. By enriching one or more desired target polynucleotides, more focus can be placed on sequencing while reducing workload and costs and/or increasing coverage depth. Examples of current enrichment schemes for next-generation sequencing include hybridization-based capture schemes, such as Agilent's SureSelect Hybrid Capture and Illumina's TruSeq Capture. Other examples include PCR-based schemes, such as Agilent's HaloPlex; ThermoFisher's AmpliSeq; Illumina's TruSeq Amplicon; and Raindance's emulsion/digital PCR.

In some embodiments, a library of polynucleotides having universal adapters at both ends is amplified using methods such as PCR. Target-specific primers containing custom adapters can be added to the reaction to amplify the target sequence. In such an embodiment, two fragment pools are generated: (a) a fragment pool having universal adapters at both ends, and (b) a fragment pool generated by selective amplification with sequence-specific adapters at one or both ends. If desired, target enrichment can be performed on the mixed pool of fragments.

In some embodiments of the methods, compositions and kits of the invention, more than one target-specific primer is employed or provided for amplification. Amplification can be single or multiplex. Multiplex PCR is a molecular biology technique for amplifying multiple nucleic acid targets in a single PCR experiment. Kits for multiplex amplification of target sequences can be obtained from Multiplicom NV.

In some embodiments of the methods, compositions and kits of the present invention, polynucleotide amplicons are used in transposable element (TE) schemes. By using a transposase to insert a transposon comprising an adapter, the adapter can be attached to the amplicon, thereby providing an adapter at the end of the amplicon fragment. In some embodiments, the polynucleotide can be fragmented and barcoded simultaneously. For example, a transposase (e.g., NEXTERA) can be used to fragment the polynucleotide and add a barcode to the polynucleotide.

Fusion gene

The target-specific primer may be complementary or identical to a portion of any known or suspected fusion gene. For example, the target-specific primer may be complementary or identical to any fusion gene disclosed in US20100279890, US20140120540, US20140272956, or US20140315199. As a further example, the target-specific primer may be complementary or identical to any of the following fusion genes: BCR-ABL, EML4–ALK, TEL-AML1, AML1-ETO, and TMPRSS2-ERG. Alternatively, the target-specific primer may be complementary or identical to a newly discovered fusion gene or the junction of such a fusion gene. Alternatively, the target-specific primer may be complementary or identical to a suspected or hypothetical fusion gene or the junction of such a fusion gene.

In some embodiments, the methods, compositions and kits of the present invention include multiple target-specific primers for different fusion genes. For example, multiple target-specific primers may include a first target-specific primer for a BCR-ABL junction and a second target-specific primer for EML4-ALK. In some embodiments, the methods, compositions and kits of the present invention include multiple target-specific primers for a single fusion gene (including multiple junctions for a single fusion gene). For example, multiple target-specific primers may include a first target-specific primer for a first EML4-ALK junction and a second target-specific primer for a second EML4-ALK junction. The methods, compositions and kits of the present invention may include a third target-specific primer, a fourth target-specific primer, a fifth target-specific primer, up to the twentieth target-specific primer, or even more target-specific primers.

Sequencing of target sequences

After amplification, the polynucleotide amplicon with adapters can be sequenced. For example, sequencing can be performed by extending the first primer and the second primer of the polynucleotide amplicon with adapters generated during the amplification process. In some embodiments, the first and second primer extensions are performed in the same direction on a separate amplicon or a group of identical amplicon. The first primer extension determines sequencing by detecting the bases incorporated due to the extension of the first primer (and other primers), thereby allowing at least a portion of the target sequence of the polynucleotide to be determined, particularly those located at the 5' of the adapter. The polynucleotide with adapters may include a sequencing initiation site, such as a P5 or P7 initiation site. In some embodiments, the first primer extension can also be used to detect the sequence of the adapter barcode. The second primer extension determines sequencing by detecting the bases incorporated due to the extension of the second primer, thereby allowing detection of target-specific barcodes. The sequencing of target-specific barcodes is used to confirm the presence and/or position of genes or other polynucleotides specific to the target-specific barcode in the polynucleotide of interest.

In some embodiments, sequencing is performed by massively parallel sequencing using sequencing by synthesis using reversible dye terminators. In some embodiments, sequencing is performed by massively parallel sequencing using sequencing by ligation. In some embodiments, sequencing is performed by single molecule sequencing. In some embodiments, sequencing is performed using pyrophosphate sequencing.

Any suitable reaction method can be used to sequence the polynucleotide. In some embodiments, a single reaction cycle can be completed using a single nucleotide (i.e., a nucleotide corresponding to G, A, T or C), and the method involves detecting whether a nucleotide is incorporated. If a nucleotide is incorporated, the identity of the nucleotide will become known. In such an embodiment, the method may involve cycling through all four nucleotides (i.e., nucleotides corresponding to G, A, T and C) in sequence, and one of the nucleotides should be incorporated. In such an embodiment, the addition of nucleotides can be detected by, for example, detecting pyrophosphate release, proton release or fluorescence, for which these methods are known. For example, in some embodiments, the chain terminator nucleotide can be a fluorescent nucleotide (i.e., a nucleotide with a fluorophore attached to a terminal phosphate) labeled with a terminal phosphate, and the identification step includes reading fluorescence. In other embodiments, the chain terminator nucleotide can be a fluorescent nucleotide of a quencher included on a terminal phosphate. In such an embodiment, the incorporation of nucleotides removes the quencher from the nucleotide, thereby allowing detection of fluorescent markers. In other embodiments, the terminal phosphate-labeled chain terminator nucleotide can be labeled on the terminal phosphate with a mass tag, charge label, charge blocking label, chemiluminescent label, redox label, or other detectable label.

In some embodiments, a single reaction cycle can be performed using all four nucleotides (i.e., nucleotides corresponding to G, A, T, and C), wherein each nucleotide is labeled with a different fluorophore. In such embodiments, the sequencing step can include adding four chain terminators corresponding to G, A, T, and C to the amplified polynucleotide, wherein the four chain terminators comprise different fluorophores. In such embodiments, the identifying step can include identifying which of the four chain terminators was added to the end of the primer.

The sequencing step can be performed using single-end sequencing, i.e., the first primer extension sequence and the second primer extension sequence are read in the same direction. In some embodiments, a single-end genome analyzer is enabled for sequencing the polynucleotides. In some embodiments, the method includes continuously monitoring the sequencing reaction (i.e., base incorporation) in real time. This can be achieved simply by including a "detection enzyme" in the chain extension reaction mixture and performing chain extension and detection or signal generation, reaction simultaneously. In some embodiments, as a first reaction step, a chain extension reaction is first performed separately, and then a separate "detection" reaction is performed, wherein the primer extension product is subsequently detected.

Sequencing data analysis

The genome rearrangement can be identified based on the data generated from the sequencing of the first primer extension and the second primer extension. The method of the present invention includes identifying the genome rearrangement in the polynucleotide based on the data generated from the sequencing of the first primer extension and the second primer extension. The sequencing data from the first primer extension provides the base pair sequence of the target sequence. The sequencing data from the second primer extension provides the base pair sequence of the adapter, which can be used to indicate or confirm the presence of the target sequence because the adapter is designed to specifically hybridize with the target sequence in the polynucleotide sample. The combined data provided by the two primer extensions provides positional information for determining any genome rearrangement in the polynucleotide.

The data generated from the first and second primer extensions are compared to a reference sample. Any differences between the reference sample and the data generated from the first and second primer extensions indicate that a genomic rearrangement may be present in the sample being studied. The sequence of the reference sample and the sequence generated by the first primer extension and the second primer extension relative to the reference sample can be used to identify the type and location of any genomic rearrangement.

The methods, compositions and kits of the invention can be used to detect any sequence of interest, including sequences associated with common deletion syndromes.

Example 1

Figure 1 shows a method for preparing a polynucleotide for sequencing by attaching an adapter and a barcode to a polynucleotide, as well as a polynucleotide with an adapter and a polynucleotide amplicon with an adapter generated by the present technology. According to one embodiment of the present invention, the polynucleotide with an adapter can be used to detect a fusion event using selective gene amplification. In Figure 1, the polynucleotide with an adapter 102 comprises a nucleic acid of interest, in this case, a connection point for a fusion gene. The polynucleotide with an adapter 102 comprises a first gene 104 and a second gene 106. The polynucleotide with an adapter 102 also comprises adapters 108 and 110 at each end. The adapters can be attached by any suitable procedure, such as by connection. At least one of the adapters comprises an adapter barcode 112, which can be a molecular barcode or a sample barcode.

In time period A, polynucleotides with adapters are prepared for target-specific amplification. Polynucleotides with adapters can be denatured to provide single-stranded polynucleotides, or double-stranded polynucleotides can be provided for amplification. In some embodiments, polynucleotides with adapters are amplified in a non-specific manner (e.g., by amplifying polynucleotides with adapters with primers complementary to the priming sites on the adapters of library members attached to polynucleotides with adapters. In some embodiments, as discussed above, polynucleotides with adapters are typically enriched before amplifying polynucleotides with adapters.

The adaptor-bearing polynucleotide is prepared and contacted with a first amplification primer 114 comprising a target-specific primer 116. The target-specific primer 116 is complementary to a sequence known or suspected to be present in the adaptor-bearing polynucleotide (e.g., a sequence within the second gene 106). The first amplification primer 114 also comprises a target-specific barcode 118 that is specific to a portion of a gene or other target known or suspected to be present in the sample being analyzed or the polynucleotide of interest. In this context, gene-specific does not mean that it is complementary to a gene, but rather that the barcode is specifically associated with a gene, so that detection of the sequence of the gene-specific barcode reliably indicates that the associated sequence is present.

In time period B, the polynucleotide with adapters is amplified in the presence of the first amplification primer 114 and the second amplification primer 120 to generate a library of polynucleotide amplicons with adapters. The polynucleotide amplicons with adapters contain nucleic acids of interest, adapters or their complements, and gene-specific barcodes or their complements. For ease of illustration, FIG. 1 shows a set of first amplification primers 114 and second amplification primers 120, although a large number of target-specific primers can be used for various sequences of the amplification reaction, and a large number of amplicons of nucleic acids of interest can be generated. In some embodiments, polynucleotides with adapters are enriched from a polynucleotide pool, for example where the label includes biotin or another binding partner.

In some embodiments (which may be in addition to enrichment or replace enrichment), polynucleotides (including polynucleotides with adapters) may be amplified with external or internal primers or nested primers. In such embodiments, external primers or primers used in earlier amplification rounds are target-specific primers, which do not necessarily include target-specific barcodes. Internal primers or primers for subsequent rounds of amplification are also target-specific primers, and they contain target-specific barcodes. Typically, nested PCR refers to one or more rounds of subsequent PCR amplifications performed using one or more new primers, which are internally bound to primers used in earlier rounds by at least one base pair. Nested PCR reduces the number of unwanted amplification targets by amplifying only the amplification product from the previous one with the correct internal sequence in subsequent reactions. Nested PCR typically requires the design of primers that are completely inside the previous external primer binding site.

The polynucleotide amplicon with the adapter can then be sequenced. In some embodiments, a first sequencing primer 122 complementary to the first priming site 124 of the adapter 108 is used to perform a first primer extension to sequence at least the first gene 104. Labeled nucleotides are added to the primer in the sequencing reaction, and a first extension sequence 126 complementary to the polynucleotide amplicon with the adapter is generated, thereby providing sequence information about the polynucleotide with the adapter. The first primer extension occurs at the first position of the polynucleotide amplicon with the adapter. The first priming site 124 can usually be at the 5' or 3' of the adapter barcode, depending on whether it is desired to sequence the adapter barcode together with the first gene 104 or separately from the first gene 104. A second primer extension is performed using a second sequencing primer 128 to sequence at least the gene-specific barcode 118. The second sequencing primer 128 is complementary to a portion 130 of the first amplification primer 114, which is 3' of the gene-specific barcode 118 and 5' of the target-specific sequence 116. The labeled nucleotides are added to the primers in the sequencing reaction, and a second extended sequence 132 complementary to the gene-specific barcode is generated, thereby providing sequence information about the gene-specific barcode. As described above, the ordinal numbers first and second do not mean that the first primer is used before the second primer; rather, they are used to distinguish different primers from each other.

In time period C, the data from the sequencing reaction is processed and interpreted. In some embodiments, the first extended sequence 126 is determined to be the sequence of the first gene, and the second extended sequence 132 is determined to be the sequence of a gene-specific barcode associated with the second gene. Based on these determinations, the data is interpreted as indicating the presence of a fusion gene in the nucleic acid of interest. The fusion gene contains multiple portions of the first gene and the second gene, and its presence can be determined even without directly sequencing the second gene 106 itself.

Example 2

What Fig. 2 shows is that polynucleotide can be amplified with target-specific primers in the case of early attachment with or without adapter.Polynucleotide for sequencing can be prepared as follows: adapter is attached to polynucleotide, then target-specific (workflow on the left side of Fig. 2), or there are also polynucleotide with adapter and polynucleotide amplicon with adapter generated by this technology.According to one embodiment of the present invention, the polynucleotide with adapter can be used for detecting genome rearrangement or other fusion events using selective gene amplification.In Fig. 2, polynucleotide 202 comprises nucleic acid of interest, in this case the junction point of fusion gene.Polynucleotide 102 comprises first gene 204 and second gene 206.

In time period A, polynucleotides are prepared for target-specific amplification. The polynucleotides can be denatured to provide single-stranded polynucleotides, or double-stranded polynucleotides can be provided for amplification. In some embodiments, the polynucleotides are amplified in a non-specific manner (e.g., by amplifying the polynucleotides with primers complementary to the priming sites on the adapters of the library members attached to the polynucleotides with adapters. In some embodiments, as discussed above, the polynucleotides are typically enriched before amplifying the polynucleotides.

The polynucleotide is prepared and contacted with a first amplification primer 214 comprising a target-specific primer 216. The target-specific primer 216 is complementary to a sequence known or suspected to be present in the polynucleotide, such as a sequence within the gene 206. The first amplification primer 214 also comprises a gene-specific barcode 218, which is specific to a portion of a gene known or suspected to be present in the analyzed sample or the polynucleotide of interest. The polynucleotide is also prepared and contacted with a second amplification primer 215 comprising a target-specific primer 217. The target-specific primer 217 is complementary to a sequence known or suspected to be present in the polynucleotide, such as a sequence within the gene 204. The second amplification primer 215 also comprises a barcode 219, such as a target-specific barcode, a sample barcode, a molecular barcode, or other barcode, or a combination of barcodes. One or both of the first and second amplification primers may comprise an adapter.

In time period B, the polynucleotide is amplified in the presence of the first amplification primer 214 and the second amplification primer 215 to generate a library of polynucleotide amplicons. The polynucleotide amplicons contain the nucleic acid of interest and the target-specific barcode or its complementary sequence. The polynucleotide amplicons can be polynucleotide amplicons with adapters, wherein they contain the nucleic acid of interest, the adapter or its complementary sequence and the target-specific barcode or its complementary sequence. For ease of illustration, FIG. 2 shows a set of first amplification primers 214 and second amplification primers 215, although the amplification reaction can use a large number of target-specific primers for various sequences, and a large number of amplicons of the nucleic acid of interest can be generated.

Then the polynucleotide amplicon can be ordered, or they can be subjected to other processing steps, such as enrichment, further amplification and/or the attachment of adapter.For example, adapter can be attached to each end of amplicon so that the polynucleotide amplicon with adapter has the order-checking initiation site, and/the adapter can be attached to the solid support.In time period C, the polynucleotide amplicon has hybridized with the primer attached on the solid support, and the primer has been extended to provide the complementary sequence of the polynucleotide amplicon attached on the support.Adopt the first sequencing primer 222 complementary to the first initiation site 224 of adapter 208 to carry out the first primer extension, so that at least the first gene 204 are ordered.In sequencing reaction, the nucleotide of mark is added in the primer, and the first extension sequence 226 complementary to the polynucleotide amplicon with adapter is generated, thereby the sequence information of the polynucleotide of the relevant band adapter is provided.The first primer extension occurs in the first position of the polynucleotide amplicon with adapter. The first priming site 224 can typically be 5' or 3' of the adapter barcode, depending on whether it is desired to sequence the adapter barcode together with the first gene 204 or separately from the first gene 204. A second primer extension is performed using a second sequencing primer 228 to sequence at least the gene-specific barcode 218. The second sequencing primer 228 is complementary to a portion 230 of the first amplification primer 214, which is 3' of the gene-specific barcode 218 and 5' of the target-specific sequence 216. Labeled nucleotides are added to the primer in the sequencing reaction, and a second extension sequence 232 is generated that is complementary to the gene-specific barcode, thereby providing sequence information about the gene-specific barcode. As described above, the ordinal numbers first and second do not mean that the first primer is used before the second primer; rather, they are used to distinguish different primers from each other.

In time period C, the data from the sequencing reaction is processed and interpreted. In some embodiments, the first extended sequence 226 is determined to be the sequence of the first gene, and the second extended sequence 232 is determined to be the sequence of a gene-specific barcode associated with the second gene. Based on these determinations, the data is interpreted as indicating the presence of a fusion gene in the nucleic acid of interest. The fusion gene contains multiple portions of the first gene and the second gene, and its presence can be determined even without directly sequencing the second gene 206 itself.

Exemplary embodiments

Embodiment 1. A method for preparing a polynucleotide for sequencing by attaching a target-specific barcode, the method comprising: amplifying the polynucleotide with a first amplification primer and a second amplification primer, wherein the first amplification primer hybridizes to a first priming site of the polynucleotide and the first amplification primer comprises a target-specific barcode; wherein the amplification produces a polynucleotide amplicon, and wherein the polynucleotide amplicon comprises a sequence identical or complementary to the polynucleotide of interest and the target-specific barcode.

Embodiment 2. The method of embodiment 1, wherein the second amplification primer (1) hybridizes to a portion of a linker attached to a polynucleotide at a certain distance from the first priming site, or (2) hybridizes to a second priming site of the polynucleotide, wherein the second priming site is at a certain distance from the first priming site.

Embodiment 3. The method of embodiment 1, further comprising attaching an adaptor to the polynucleotide at a distance from the first priming site, wherein the adaptor comprises a second priming site.

Embodiment 4. The method of embodiment 3, wherein the adapter further comprises an adapter barcode, wherein the adapter barcode is a sample barcode or a molecular barcode.

Embodiment 5. The method of any one of the preceding embodiments, wherein the first priming site is part of a fusion gene and the target-specific barcode is specific for the part of the fusion gene.

Embodiment 6. The method of embodiment 5, wherein the portion of the fusion gene is the junction point of the fusion gene.

Embodiment 7. The method of any one of the preceding embodiments, wherein the polynucleotide is genomic DNA (gDNA) or complementary DNA (cDNA) derived from an RNA template.

Embodiment 8. A method according to any of the preceding embodiments, wherein the polynucleotide of interest comprises a plurality of polynucleotides of interest, and the method comprises attaching a plurality of linkers to the plurality of polynucleotides, thereby forming a plurality of linker-carrying polynucleotides, wherein each of the plurality of linker-carrying polynucleotides comprises a different molecular barcode.

Embodiment 9. A method according to any one of the preceding embodiments, wherein the polynucleotide of interest comprises a plurality of polynucleotides of interest, and the first amplification primer comprises a plurality of first amplification primers having different target-specific primers and different target-specific barcodes, thereby forming a plurality of polynucleotide amplicons with adapters, wherein each of the plurality of polynucleotide amplicons with adapters comprises a different target-specific barcode.

Embodiment 10. The method of any one of the preceding embodiments, wherein the polynucleotide amplicon or polynucleotide with adaptor comprises a binding partner, such as a biotin moiety.

Embodiment 11. The method of any of the preceding embodiments, further comprising sequencing the polynucleotide amplicon at the first and second positions by performing a first primer extension and a second primer extension, wherein the first primer extension and the second primer extension are performed in the same direction. Sequencing at the first position can provide a sequence of at least a portion of the polynucleotide of interest, and sequencing at the second position can provide a sequence of a target-specific barcode.

Embodiment 12. The method of embodiment 11, wherein the first primer extension and the second primer extension are performed in the same direction on the polynucleotide in separate sequencing runs.

Embodiment 13. The method of embodiment 11, wherein the sequencing is next generation sequencing (NGS) or massively parallel sequencing.

Embodiment 14. The method of any of the preceding embodiments, further comprising detecting genomic rearrangements using single-end sequencing of at least one of the polynucleotide amplicons, such as by identifying genomic rearrangements based on data generated from sequencing of first primer extension and second primer extension.

Embodiment 15. The method of embodiment 14, wherein the frequency of genomic rearrangement is about 10% or less, or 5% or less.

Embodiment 16. The method of embodiment 14, wherein the genomic rearrangement is a translocation.

Embodiment 17. The method of embodiment 14, wherein the data generated from sequencing extended from the first primer is compared to a known nucleic acid sequence, such as a known gDNA sequence, to determine the genomic rearrangement.

Embodiment 18. A method for preparing a library of polynucleotides for sequencing by attaching target-specific barcodes, the method comprising: amplifying a polynucleotide pool using a first set of amplification primers and a second set of amplification primers, wherein the first set of amplification primers hybridizes to a plurality of different sequences within the polynucleotide pool, wherein each of the first set of amplification primers comprises a different target-specific barcode.

Embodiment 19. The method of embodiment 18, further comprising: generating a library of polynucleotides with adapters, wherein each polynucleotide with adapters comprises an adapter attached to the polynucleotide, and the adapter comprises a second priming site and an adapter barcode.

Embodiment 20. The method of embodiment 19, wherein the second set of amplification primers hybridizes to a second priming site on the adaptor, thereby generating a polynucleotide amplicon with an adaptor.

Embodiment 21. The method of embodiment 18, further comprising sequencing each of a plurality of polynucleotide amplicons with adapters at two positions by performing a first primer extension and a second primer extension, wherein for each of the polynucleotide amplicons, sequencing by the first primer extension and the second primer extension is performed in the same direction.

Embodiment 22. The method of embodiment 21 further comprises: identifying genomic rearrangements based on data generated from sequencing of the first primer extension and the second primer extension.

Embodiment 23. A composition or kit for detecting genomic rearrangements in a polynucleotide having a first binding site, the composition or kit comprising: a first amplification primer comprising a target-specific primer and a target-specific barcode; and a second amplification primer.

Embodiment 24. The composition or kit of embodiment 23, further comprising: an adaptor comprising a second priming site and an adaptor barcode, and wherein the second amplification primer comprises a priming sequence that is complementary to or identical to a sequence within the adaptor.

Embodiment 25. A composition or kit of embodiment 23, wherein the second amplification primer (1) hybridizes to a portion of an adapter attached to a polynucleotide at a certain distance from the first priming site, or (2) hybridizes to a second priming site of the polynucleotide, wherein the second priming site is at a certain distance from the first priming site.

Embodiment 26. The composition or kit of embodiment 24, wherein the adaptor and/or the second priming site is at the 5' end of the strand of the polynucleotide and the first priming site is at the 3' end of the strand.

Embodiment 27. A method for detecting genomic rearrangements in a polynucleotide, the method comprising: amplifying the polynucleotide using a first amplification primer and a second amplification primer, wherein the first amplification primer hybridizes to a first priming site of the polynucleotide and the first amplification primer further comprises a target-specific barcode, wherein the amplification produces a polynucleotide amplicon comprising a sequence identical or complementary to the polynucleotide of interest and the target-specific barcode; and sequencing the polynucleotide amplicon at a first and a second position by performing a first primer extension and a second primer extension, wherein the first primer extension and the second primer extension are performed in the same direction.

Embodiment 28. The method of embodiment 27, wherein sequencing at the first position provides a sequence of at least a portion of the polynucleotide of interest and sequencing at the second position provides a sequence of a target-specific barcode.

Embodiment 29. The method of embodiment 27 or 28, wherein the first primer extension and the second primer extension are performed in the same direction on the polynucleotide during separate sequencing runs.

Embodiment 30. The method of any one of embodiments 27 to 39, wherein the sequencing is next generation sequencing (NGS) or massively parallel sequencing.

Embodiment 31. The method of any one of embodiments 27 to 30, further comprising detecting a genomic rearrangement using single-end sequencing of at least one of the polynucleotide amplicons, such as by identifying the genomic rearrangement based on data generated from sequencing of a first primer extension and a second primer extension.

Embodiment 32. The method of embodiment 31, wherein the frequency of genomic rearrangement is about 10% or less.

Embodiment 33. The method of embodiment 31 or 32, wherein the genomic rearrangement is a translocation.

Embodiment 34. The method of any one of embodiments 27 to 33, wherein the data generated from sequencing extended from the first primer is compared to a known nucleic acid sequence, such as a known gDNA sequence, to determine genomic rearrangements.

In view of this disclosure, it should be noted that the method can be implemented in accordance with the present teachings. In addition, various components, materials, structures and parameters are included for illustration and example only, without any restrictive meaning. In view of this disclosure, the present teachings can be implemented in other applications, and the components, materials, structures and equipment for implementing these applications can be determined while remaining within the scope of the appended claims.

Claims (20)

1. A method of preparing a polynucleotide for sequencing by attaching a target-specific barcode, the method comprising:

Amplifying a polynucleotide with a first amplification primer and a second amplification primer, wherein the first amplification primer hybridizes to a first priming site of the polynucleotide and the first amplification primer comprises a target-specific barcode, wherein the second amplification primer hybridizes to (1) a portion of an adapter attached to the polynucleotide at a distance from the first priming site, or (2) a second priming site of the polynucleotide, wherein the second priming site hybridizes to a second priming site at a distance from the first priming site, wherein the first priming site is a portion of a fusion gene and the target-specific barcode is specific for the portion of the fusion gene;

wherein said amplification produces a polynucleotide amplicon, wherein the polynucleotide amplicon comprises sequences identical or complementary to the polynucleotide of interest and the target-specific barcode,

Wherein the method further comprises single-ended sequencing of the polynucleotide amplicon at the first and second locations by performing a first sequencing primer extension and a second sequencing primer extension, wherein the first sequencing primer extension and the second sequencing primer extension are performed in the same direction.

2. The method of claim 1, further comprising attaching an adapter to the polynucleotide at a distance from a first priming site, wherein the adapter comprises a second priming site.

3. The method of claim 1, wherein the portion of the fusion gene is the junction of the fusion gene.

4. The method of claim 1, wherein the polynucleotide of interest comprises a plurality of polynucleotides of interest, and the method comprises attaching a plurality of adaptors to the plurality of polynucleotides, thereby forming a plurality of adaptor-containing polynucleotides each comprising a different molecular barcode.

5. The method of claim 1, wherein the polynucleotide of interest comprises a plurality of polynucleotides of interest and the first amplification primer comprises a plurality of first amplification primers having different target-specific primers and different target-specific barcodes, thereby forming a plurality of adaptor-containing polynucleotide amplicons, wherein each of the plurality of adaptor-containing polynucleotide amplicons comprises a different target-specific barcode.

6. The method of claim 1, wherein the polynucleotide amplicon or the adaptor-bearing polynucleotide comprises a binding partner.

7. The method of claim 6, wherein the binding partner is a biotin moiety.

8. The method of claim 1, wherein first sequencing primer extension and second sequencing primer extension are performed in the same direction on the polynucleotide in separate sequencing runs.

9. A composition or kit for detecting genomic rearrangement in a polynucleotide having a first priming site by single ended sequencing, the composition or kit comprising:

A first amplification primer comprising a target-specific primer and a target-specific barcode; and

A second amplification primer comprising a second primer sequence,

Wherein the first priming site is a portion of a fusion gene and the target-specific barcode is specific for the portion of the fusion gene,

Wherein the second amplification primer hybridizes (1) to a portion of an adapter attached to the polynucleotide at a distance from the first priming site, or (2) to a second priming site of a polynucleotide,

Wherein the single-ended sequencing comprises performing a first sequencing primer extension and performing a second sequencing primer extension, wherein the first sequencing primer extension and the second sequencing primer extension are performed in the same direction.

10. The composition or kit of claim 9, further comprising:

an adapter comprising a second priming site and an adapter barcode, and

Wherein the second amplification primer comprises a priming sequence that is complementary to or identical to a sequence within the adapter.

11. A method of detecting genomic rearrangements in a polynucleotide, the method comprising:

Amplifying a polynucleotide with a first amplification primer and a second amplification primer, wherein the first amplification primer hybridizes to a first priming site of the polynucleotide and the first amplification primer further comprises a target-specific barcode,

Wherein the first priming site is a portion of a fusion gene and the target-specific barcode is specific for the portion of the fusion gene,

Wherein the second amplification primer hybridizes (1) to a portion of an adapter attached to the polynucleotide at a distance from the first priming site, or (2) to a second priming site of a polynucleotide,

Wherein the amplification produces a polynucleotide amplicon comprising the same or complementary sequence as the polynucleotide of interest and the target-specific barcode; and

Single-ended sequencing of the polynucleotide amplicon at the first and second locations by performing a first sequencing primer extension and a second sequencing primer extension, wherein the first sequencing primer extension and the second sequencing primer extension are performed in the same direction,

Wherein the method is not used for diagnosing a disease.

12. The method of claim 11, wherein sequencing at the first location provides a sequence of at least a portion of the polynucleotide of interest and sequencing at the second location provides a sequence of a target-specific barcode.

13. The method of claim 11, wherein first sequencing primer extension and second sequencing primer extension are performed in the same direction on the polynucleotide in separate sequencing runs.

14. The method of claim 11, wherein the sequencing is next generation sequencing or large scale parallel sequencing.

15. The method of claim 11, further comprising detecting genomic rearrangements using single ended sequencing of at least one of the polynucleotide amplicons.

16. The method of claim 15, wherein the genomic rearrangement is identified by data generated based on sequencing of the first sequencing primer extension and the second sequencing primer extension.

17. The method of claim 15, wherein the frequency of genomic rearrangements is 10% or less.

18. The method of claim 15, wherein the genomic rearrangement is translocation.

19. The method of claim 11, wherein the data generated from sequencing the first primer extension is compared to a known nucleic acid sequence to determine a genomic rearrangement.

20. The method of claim 19, wherein the known nucleic acid sequence is a known gDNA sequence.